Project Summary: The TGF-? family consists of 33 individual ligands with fundamental roles throughout human biology. Due to their biological importance, multiple forms of regulation exist to control ligand signaling, many of which are specific for a subset of ligands. This project will focus on the signaling and regulation of two closely related TGF-? ligands - Growth and Differentiation Factor 8 (GDF8) and GDF11. Functionally, GDF8 is a strong negative regulator of muscle mass. As such, inhibitors to GDF8 have been pursued to therapeutically induce muscle hypertrophy in order to treat muscle-wasting pathologies. On the other hand, GDF11, which shares 90% identity to GDF8, has a role in erythrocyte differentiation where its inhibition has been shown to boost red blood cell production. Both GDF8 and GDF11 signal through similar receptors and are controlled by a combination of latency and extracellular antagonists. However, we have limited information regarding the mechanisms and molecular details that regulate GDF8 and GDF11, in part due to a lack of the structural information describing these interactions. The overall goal and long-term objective is to understand the molecular mechanisms that regulate GDF8 and GDF11 signaling. We will take a structure-function approach, coupling structural studies (X- ray crystallography and NMR) with binding analysis and in vitro cell-based assays. Observations will be extended to in vivo, in mouse models using recombinant GDF8/11 complexes. We will pursue three specific aims which will involve (1) resolving the molecular details of GDF8 and GDF11 with its cognate receptors, (2) characterizing the latent state of GDF8 and GDF11 and deciphering how they are activated from latency and (3) determining the structural mechanism of the extracellular antagonist GASP and determining how antagonism is specific for GDF8 and GDF11. Collectively, these aims will provide a deeper understanding of the mechanism that regulate GDF8 and GDF11 signaling, which can ultimately be used to facilitate or augment current therapeutic efforts to modulate ligand signaling.